Prolyl tRNA synthetase polynucleotides

ABSTRACT

The invention provides proS polypeptides and DNA (RNA) encoding proS polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing proS polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This application claims the benefit of UK application number 9607991.8,filed Apr. 18, 1996.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the prolyl tRNA synthetase family, hereinafter referredto as "proS".

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes knownto cause several types of disease in humans, including, for example,otitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid. Sinceits isolation more than 100 years ago, Streptococcus pneumoniae has beenone of the more intensively studied microbes. For example, much of ourearly understanding that DNA is, in fact, the genetic material waspredicated on the work of Griffith and of Avery, Macleod and McCartyusing this microbe. Despite the vast amount of research with S.pneumoniae, many questions concerning the virulence of this microberemain. It is particularly preferred to employ Streptococcal genes andgene products as targets for the development of antibiotics.

The frequency of Streptococcus pneumoniae infections has risendramatically in the past 20 years. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Streptococcus pneumoniae strains which are resistantto some or all of the standard antibiotics. This has created a demandfor both new anti-microbial agents and diagnostic tests for thisorganism.

The t-RNA synthetases have a primary role in protein synthesis accordingto the following scheme:

    Enzyme+ATP+AA⃡Enzyme.AA-AMP+PPi

    Enzyme.AA-AMP+t-RNA⃡Enzyme+AMP+AA-t-RNA

in which AA is an amino acid.

Inhibition of this process leads to a reduction in the levels of chargedt-RNA and this triggers a cascade of responses known as the stringentresponse, the result of which is the induction of a state of dormancy inthe organism. As such selective inhibitors of bacterial t-RNA synthetasehave potential as antibacterial agents. One example of such is mupirocinwhich is a selective inhibitor of isoleucyl t-RNA synthetase. Othert-RNA synthetases are now being examined as possible anti-bacterialtargets, this process being greatly assisted by the isolation of thesynthetase.

Clearly, there is a need for factors, such as the novel compounds of theinvention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists which can play a role inpreventing, ameliorating or correcting infections, dysfunctions ordiseases.

The polypeptides of the invention have amino acid sequence homology to aknown Escherichia coli prolyl tRNA synthetase protein.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel proS polypeptides by homology between an amino acidsequence set out in Table 1 SEQ ID NO: 2! and a known amino acidsequence or sequences of other proteins such as Escherichia coli prolyltRNA synthetase protein.

It is a further object of the invention to provide polynucleotides thatencode proS polypeptides, particularly polynucleotides that encode thepolypeptide herein designated proS.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding proS polypeptides comprisinga sequence set out in Table 1 SEQ ID NO:1, 3!, or a variant thereof.

In another particularly preferred embodiment of the invention there is anovel proS protein from Streptococcus pneumoniae comprising an aminoacid sequence of Table 1 SEQ ID NO:2, 4!, or a variant thereof.

In accordance with another aspect of the invention there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Streptococcus pneumoniae 0100993 strain contained in thedeposited strain.

A further aspect of the invention there are provided isolated nucleicacid molecules encoding proS, particularly Streptococcus pneumoniaeproS, including mRNAs, cDNAs, genomic DNAs. Further embodiments of theinvention include biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of proS and polypeptides encoded thereby.

Another aspect of the invention there are provided novel polypeptides ofStreptococcus pneumoniae referred to herein as proS as well asbiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of proS polypeptide encoded by naturally occurring alleles ofthe proS gene.

In a preferred embodiment of the invention there are provided methodsfor producing the aforementioned proS polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing proSexpression, treating disease, for example, otitis media, conjunctivitis,pneumonia, bacteremia, meningitis, sinusitis, pleural empyema andendocarditis, and most particularly meningitis, such as for exampleinfection of cerebrospinal fluid, assaying genetic variation, andadministering a proS polypeptide or polynucleotide to an organism toraise an immunological response against a bacteria, especially aStreptococcus pneumoniae bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to proS polynucleotide sequences, particularly under stringentconditions.

In certain preferred embodiments of the invention there are providedantibodies against proS polypeptides.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided proS agonists and antagonists, preferably bacteriostatic orbacteriocidal agonists and antagonists.

In a further aspect of the invention there are provided compositionscomprising a proS polynucleotide or a proS polypeptide foradministration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"Host cell" is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, "identity" also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. "Identity" and "similarity" can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). As an illustration, by a polynucleotide having anucleotide sequence having at least, for example, 95% "identity" to areference nucleotide sequence of SEQ ID NO: 1 it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence of SEQ ID NO: 1. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5 or 3 terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously , by a polypeptide having an amino acidsequence having, at least, for example, 95% identity to a referenceamino acid sequence of SEQ ID NO:2 is intended that the amino acidsequence of the polypeptide is identical to the reference sequenceexcept that the polypeptide sequence may include up to five amino acidalterations per each 100 amino acids of the reference amino acid of SEQID NO: 2. In other words, to obtain a polypeptide having, an amino acidsequence at least 95% identical to a reference amino acid sequence, upto 5% of the amino acid residues in the reference sequence may bedeleted or substituted with another amino acid, or a number of aminoacids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among, residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

"Isolated" means altered "by the hand of man" from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not "isolated,"but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is "isolated", as the term is employedherein.

"Polynucleotide(s)" generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotide(s)" include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, "polynucleotide" as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term "polynucleotide(s)" also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are"polynucleotide(s)" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term"polynucleotide(s)" as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells."Polynucleotide(s)" also embraces short polynucleotides often referredto as oligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. "Polypeptide(s)" refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. "Polypeptide(s)" includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

"Variant(s)" as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

The invention relates to novel proS polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a novel proS of Streptococcuspneumoniae, which is related by amino acid sequence homology toEscherichia coli prolyl tRNA synthetase polypeptide. The inventionrelates especially to proS having the nucleotide and amino acidsequences set out in Table 1 SEQ ID NO: 1, 3! and Table 1 SEQ ID NO: 2,4! respectively, and to the proS nucleotide sequences of the DNA in thedeposited strain and amino acid sequences encoded thereby.

                                      TABLE 1    __________________________________________________________________________    proS Polynucleotide and Polypeptide Sequences    __________________________________________________________________________    (A) Sequences from Streptococcus pneumoniae proS polynucleotide    sequence  SEQ ID NO:1!.    5'-1 ATGAAACAAA GTAAAATGCC TATCCCAACG CTTCGCGAAA TGCCAAGCGA         51  TGCTCAAGTT ATCAGCCATG CTCTTATGTT GCGTGCTGGT TATGTTCGCC        101  AAGTTTCAGC AGGTGTTTAT TCTTATCTAC CACTTGCCAA CCGTGTGATT        151  GAAAAAGCTA AAAACATCAT GCGCCAAGAA TTCGAAAAGA TTGGTGCTGT        201  TGAGATGTTG GCTCCAGCCC TTCTTAGTGC AGAATTGTGG CGTGAATCAG        251  GTCGTTACGA AACCTATGGT GAAGACCTTT ACAAACTGAA AAACCGTGAA        301  AAATCAGACT TTATCTTAGG TCCAACTCAC GAAGAAACCT TTACAGCTAT        351  TGTCCGTGAT TCTGTTAAAT CTTACAAGCA ATTGCCACTC AACCTTTATC        401  AAATTCAGCC CAAGTATCGT GATGAAAAAC GCCCACGTAA TGGACTTCTT        451  CGTACACGTG AGTTTATCAT GAAGGATGCT TATAGTTTCC ACGCTAACTA        501  TGATAGTTTG GATAGTGTTT ATGATGAGTA CAAAGCAGCC TATGAGCGTA        551  TTTTCACTCG TAGTGGTTTA GACTTCAAGG CTATTATTGG TGACGGTGGA        601  GCCATGGGTG GTAAGGATAG CCAAGAATTT ATGGCCATTA GGCCTATCTG        651  CCGTACAGAC CTTGACCGCT GGGTTGTCTT GGACAAGTCA GTTGCCTCAT        701  TTGACGAAAT TCCTGCAGAA GTGCAAGAAG AAATCAAGGC AGAATTGCTC        751  AAATGGATAG TCTCTGGTGA AGATACCATT GCTTACTCAA GTGAGTCTAG        801  CTATGCAGCT AACTTAGAAA TGGCAACAAA CGAGTACAAA CCAAGCAACC        851  GTGTTGTCGC TGAAGAAGAA GTTACTCGTG TTGAAACGCC AGATGTTAAA        901  TCAATTGATG AAGTTGCAGC CTTCCTCAAT GTTCCAGAAG AACAAACGAT        951  TAAAACCCTC TTCTACATTG CAGATGGTGA GCTTGTTGCA GCCCTTCTAG       1001 TTGGAAATGA CCAACTCAAC GAAGTCAAGT TGAAAAATCA CTTGGGAGCA       1051 AATTTCTTTG ACGTTGCTAG CGAAGAAGAA GTGGCGAATG TTGTTCAAGC       1101 AGGATTTGGT TCACTTGGAC CAGTTGGTTT GCCAGAGAAT ATTAAAATTA       1151 TTGCAGATCG TAAGGTGCAA GATGTTCGCA ATGCAGTTGT CGGTGCTAAC       1201 GAAGATGGCT ACCACTTGAC TGGTGTGAAC CCAGGCCGTG ATTTTACTGC       1251 AGAATATGTG GATATCCGTG AAGTTCGTGA GGGTGAAATT TCCCCAGATG       1301 GACAAGGTGT CCTTAACTTT GCGCGTGGTA TTGAGATCGG TCATATTTTC       1351 AAACTCGGAA CTCGCTATTC AGCAAGCATG GGAGCAGATG TCTTGGATGA       1401 AAATGGTCGT GCTGTGCCAA TCATCATGGG ATGTTACGGT ATCGGTGTCA       1451 GCCGTCTTCT TTCAGCAGTG ATGGAGCAAC ACGCTCGCCT CTTTGTTAAC       1501 AAAACGCCAA AAGGTGAATA CCGTTACGCT TGGGGAATCA ATTTCCCTAA       1551 AGAATTGGCA CCATTTGATG TGCATTTGAT TACTGTTAAT GTCAAGGATG       1601 AAGAAGCGCA AGCCTTGACA GAAAAACTTG AAGCAAGCTT GATGGGAGAT       1651 C-3'    (B) proS polypeptide sequence deduced from the polynucleotide    sequence in this table  SEQ ID NO:2!.    NH.sub.2 -1  MKQSKMPIPT LREMPSDAQV ISHALMLRAG YVRQVSAGVY SYLPLANRVI         51 EKAKNIMRQE FEKIGAVEML APALLSAELW RESGRYETYG EDLYKLKNRE        101 KSDFILGPTH EETFTAIVRD SVKSYKQLPL NLYQIQPKYR DEKRPRNGLL        151 RTREFIMKDA YSFHANYDSL DSVYDEYKAA YERIFTRSGL DFKAIIGDGG        201 AMGGKDSQEF MAIRPICRTD LDRWVVLDKS VASFDEIPAE VQEEIKAELL        251 KWIVSGEDTI AYSSESSYAA NLEMATNEYK PSNRVVAEEE VTRVETPDVK        301 SIDEVAAFLN VPEEQTIKTL FYIADGELVA ALLVGNDQLN EVKLKNHLGA        351 NFFDVASEEE VANVVQAGFG SLGPVGLPEN IKIIADRKVQ DVRNAVVGAN        401 EDGYHLTGVN PGRDFTAEYV DIREVREGEI SPDGQGVLNF ARGIEIGHIF        451 KLGTRYSASM GADVLDENGR AVPIIMGCYG IGVSRLLSAV MEQHARLFVN        501 KTPKGEYRYA WGINFPKELA PFDVHLITVN VKDEEAQALT EKLEASLMGD-COOH    (C) Polynucleotide sequence embodiments  SEQ ID NO:1!.    X-(R.sub.1).sub.n -1 ATGAAACAAA GTAAAATGCC TATCCCAACG CTTCGCGAAA TGCCAAGCG           51  TGCTCAAGTT ATCAGCCATG CTCTTATGTT GCGTGCTGGT TATGTTCGCC          101 AAGTTTCAGC AGGTGTTTAT TCTTATCTAC CACTTGCCAA CCGTGTGATT          151 GAAAAAGCTA AAAACATCAT GCGCCAAGAA TTCGAAAAGA TTGGTGCTGT          201 TGAGATGTTG GCTCCAGCCC TTCTTAGTGC AGAATTGTGG CGTGAATCAG          251 GTCGTTACGA AACCTATGGT GAAGACCTTT ACAAACTGAA AAACCGTGAA          301 AAATCAGACT TTATCTTAGG TCCAACTCAC GAAGAAACCT TTACAGCTAT          351 TGTCCGTGAT TCTGTTAAAT CTTACAAGCA ATTGCCACTC AACCTTTATC          401 AAATTCAGCC CAAGTATCGT GATGAAAAAC GCCCACGTAA TGGACTTCTT          451 CGTACACGTG AGTTTATCAT GAAGGATGCT TATAGTTTCC ACGCTAACTA          501 TGATAGTTTG GATAGTGTTT ATGATGAGTA CAAAGCAGCC TATGAGCGTA          551 TTTTCACTCG TAGTGGTTTA GACTTCAAGG CTATTATTGG TGACGGTGGA          601 GCCATGGGTG GTAAGGATAG CCAAGAATTT ATGGCCATTA GGCCTATCTG          651 CCGTACAGAC CTTGACCGCT GGGTTGTCTT GGACAAGTCA GTTGCCTCAT          701 TTGACGAAAT TCCTGCAGAA GTGCAAGAAG AAATCAAGGC AGAATTGCTG          751 AAATGGATAG TCTCTGGTGA AGATACCATT GCTTACTCAA GTGAGTCTAG          801 CTATGCAGCT AACTTAGAAA TGGCAACAAA CGAGTACAAA CCAAGCAACC          851 GTGTTGTCGC TGAAGAAGAA GTTACTCGTG TTGAAACGCC AGATGTTAAA          901 TCAATTGATG AAGTTGCAGC CTTCCTCAAT GTTCCAGAAG AACAAACGAT          951 TAAAACCCTC TTCTACATTG CAGATGGTGA GCTTGTTGCA GCCCTTCTAG         1001 TTGGAAATGA CCAACTCAAC GAAGTCAAGT TGAAAAATCA CTTGGGAGCA         1051 AATTTCTTTG ACGTTGCTAG CGAAGAAGAA GTGGCGAATG TTGTTCAAGC         1101 AGGATTTGGT TCACTTGGAC CAGTTGGTTT GCCAGAGAAT ATTAAAATTA         1151 TTGCAGATCG TAAGGTGCAA GATGTTCGCA ATGCAGTTGT CGGTGCTAAC         1201 GAAGATGGCT ACCACTTGAC TGGTGTGAAC CCAGGCCGTG ATTTTACTGC         1251 AGAATATGTG GATATCCGTG AAGTTCGTGA GGGTGAAATT TCCCCAGATG         1301 GACAAGGTGT CCTTAACTTT GCGCGTGGTA TTGAGATCGG TCATATTTTC         1351 AAACTCGGAA CTCGCTATTC AGCAAGCATG GGAGCAGATG TCTTGGATGA         1401 AAATGGTCGT GCTGTGCCAA TCATCATGGG ATGTTACGGT ATCGGTGTCA         1451 GCCGTCTTCT TTCAGCAGTG ATGGAGCAAC ACGCTCGCCT CTTTGTTAAC         1501 AAAACGCCAA AAGGTGAATA CCGTTACGCT TGGGGAATCA ATTTCCCTAA         1551 AGAATTGGCA CCATTTGATG TGCATTTGAT TACTGTTAAT GTCAAGGATG         1601 AAGAAGCGCA AGCCTTGACA GAAAAACTTG AAGCAAGCTT GATGGGAGAT         1651 C-(R.sub.2).sub.n -Y    (D) Polypeptide sequence embodiments  SEQ ID NO:2!.    X-(R.sub.1).sub.n -1  MKQSKMPIPT LREMPSDAQV ISHALMLRAG YVRQVSAGVY    SYLPLANRVI           51 EKAKNIMRQE FEKIGAVEML APALLSAELW RESGRYETYG EDLYKLKNRE          101 KSDFILGPTH EETFTAIVRD SVKSYKQLPL NLYQIQPKYR DEKRPRNGLL          151 RTREFIMKDA YSFHANYDSL DSVYDEYKAA YERIFTRSGL DFKAIIGDGG          201 AMGGKDSQEF MAIRPICRTD LDRWVVLDKS VASFDEIPAE VQEEIKAELL          251 KWIVSGEDTI AYSSESSYAA NLEMATNEYK PSNRVVAEEE VTRVETPDVK          301 SIDEVAAFLN VPEEQTIKTL FYIADGELVA ALLVGNDQLN EVKLKNHLGA          351 NFFDVASEEE VANVVQAGFG SLGPVGLPEN IKIIADRKVQ DVRNAVVGAN          401 EDGYHLTGVN PGRDFTAEYV DIREVREGEI SPDGQGVLNF ARGIEIGHIF          451 KLGTRYSASM GADVLDENGR AVPIIMGCYG IGVSRLLSAV MEQHARLFVN          501 KTPKGEYRYA WGINFPKELA PFDVHLITVN VKDEEAQALT EKLEASLMGD-(R.sub.2)    .sub.n -Y    (E) Polynucleotide sequence embodiment  SEQ ID NO:3!.    5'-1 TTGAAAAAGC TAAAAAACAT CATGCGCCAA GAATTCGAAA AGATTGGTGC        51  TGTTGAGATG TTGGCTCCAG CCCTTCTTAG TGCAGAATTG TGGCGTGAAT       101  CAGGTCGTTA CGAAACGTAT GGTGAAGACC TTTACAAACT GAAAAACCGT       151  GAAAAATCAG ACTTTATCTT AGGTCCAACT CACGAAGAAA CCTTTACAGC       201  TATTGTCCGT GATTCTGTTA AATCTTACAA GCAATTGCCA CTCAACCTTT       251  ATCAAATTCA GCCCAAGTAT CGTGATGAAA AACGCCCACG TAATGGACTT       301  CTTCGTACAC GTGAGTTTAT CATGAAGGAT GCTTATAGTT TCCACGCTAA       351  CTATGATAGT TTGGATAGTG TTTATGATGA GTACAAAGCA GCCTATGAGC       401  GTATTTTCAC TCGTAGTGGT TTAGACTTCA AGGCTATTAT TGGTGACGGT       451  GGAGCCATGG GTGGTAAGGA TAGCCAAGAA TTTATGG-3'    (F) Polypeptide sequence embodiment  SEQ ID NO:4!.    NH.sub.2 -1 LKKLKNIMRQ EFEKIGAVEM LAPALLSAEL WRESGRYETY GEDLYKLKNR         51  EKSDFILGPT HEETFTAIVR DSVKSYKQLP LNLYQIQPKY RDEKRPRNGL        101  LRTREFIMKD AYSFHANYDS LDSVYDEYKA AYERIFTRSG LDFKAIIGDG        151  GAMGGKDSQE FM-COOH    __________________________________________________________________________

Deposited Materials

A deposit containing a Streptococcus pneumoniae 0100993 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Apr. 11, 1996 and assigned deposit number 40794. The depositwas described as Streptococcus pneumoniae 0100993 on deposit. On 17April 1996 a Streptococcus pneumoniae 0100993 DNA library in E. coli wassimilarly deposited with the NCIMB and assigned deposit number 40800.The Streptococcus pneumoniae strain deposit is referred to herein as"the deposited strain" or as "the DNA of the deposited strain."

The deposited strain contains the full length proS gene. The sequence ofthe polynucleotides contained in the deposited strain, as well as theamino acid sequence of the polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

Polypeptides

The polypeptides of the invention include polypeptides of Table 1 SEQ IDNO:2, 4! (in particular the mature polypeptide) as well as polypeptidesand fragments, particularly those which have the biological activity ofproS, and also those which have at least 70% identity to a polypeptideof Table 1 SEQ ID NO:2, 4! or the relevant portion, preferably at least80% identity to a polypeptide of Table 1 SEQ ID NO:2, 4!, and morepreferably at least 90% similarity (more preferably at least 90%identity) to a polypeptide of Table 1 SEQ ID NO:2, 4! and still morepreferably at least 95% similarity (still more preferably at least 95%identity) to a polypeptide of Table 1 SEQ ID NO:2, 4! and also includeportions of such polypeptides with such portion of the polypeptidegenerally containing at least 30 amino acids and more preferably atleast 50 amino acids.

The invention also includes polypeptides of the formula set forth inTable 1 (D) wherein, at the amino terminus, X is hydrogen, and at thecarboxyl terminus, Y is hydrogen or a metal, R₁ and R₂ is any amino acidresidue, and n is an integer between 1 and 1000. Any stretch of aminoacid residues denoted by either R group, where R is greater than 1, maybe either a heteropolymer or a homopolymer, preferably a heteropolymer.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with pros polypeptides fragments maybe "free-standing," or comprised within a larger polypeptide of whichthey form a part or region, most preferably as a single continuousregion, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion AN amino acid sequence of Table 1 SEQ ID NO:2, 4!, or ofvariants thereof, such as a continuous series of residues that includesthe amino terminus, or a continuous series of residues that includes thecarboxyl terminus. Degradation forms of the polypeptides of theinvention in a host cell, particularly a Streptococcus pneumoniae, arealso preferred. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of proS, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Streptococcuspneumoniae or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides thatencode the proS polypeptide having the deduced amino acid sequence ofTable 1 SEQ ID NO:2, 4! and polynucleotides closely related thereto andvariants thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 SEQ ID NO:1, 3!, a polynucleotide of the inventionencoding proS polypeptide may be obtained using standard cloning andscreening methods, such as those for cloning and sequencing chromosomalDNA fragments from bacteria using Streptococcus pneumoniae 0100993 cellsas starting material, followed by obtaining a full length clone. Forexample, to obtain a polynucleotide sequence of the invention, such as asequence given in Table 1 SEQ ID NO:1, 3!, typically a library of clonesof chromosomal DNA of Streptococcus pneumoniae 0100993 in E.coli or someother suitable host is probed with a radiolabeled oligonucleotide,preferably a 17-mer or longer, derived from a partial sequence. Clonescarrying DNA identical to that of the probe can then be distinguishedusing stringent conditions. By sequencing the individual clones thusidentified with sequencing primers designed from the original sequenceit is then possible to extend the sequence in both directions todetermine the full gene sequence. Conveniently, such sequencing isperformed using denatured double stranded DNA prepared from a plasmidclone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York(1989). (see in particular Screening By Hybridization 1.90 andSequencing Denatured Double-Stranded DNA Templates 13.70). Illustrativeof the invention, each polynucleotide set out in Table 1 SEQ ID NO:1, 3!was discovered in a DNA library derived from Streptococcus pneumoniae0100993.

The DNA sequences set out in Table 1 SEQ ID NO:1, 3! each contain anopen reading frame encoding a protein having about the number of aminoacid residues set forth in Table 1 SEQ ID NO:2, 4 respectively! with adeduced molecular weight that can be calculated using amino acid residuemolecular weight values well known in the art. The start codon of theDNA in Table 1 is nucleotide number 1 and the codon that encodes an lastamino acid is number 1648, the stop codon being the next codon followingthis last codon encoding an amino acid (nucleotides 1649-1651).

proS of the invention is structurally related to other proteins of theprolyl tRNA synthetase family, as shown by the results of sequencing theDNA encoding proS of the deposited strain. The protein exhibits greatesthomology to Escherichia coli prolyl tRNA synthetase protein among knownproteins. proS of Table 1 SEQ ID NO:2! has about 48% identity over itsentire length and about 67% similarity over its entire length with theamino acid sequence of Escherichia coli prolyl tRNA synthetasepolypeptide.

The invention provides a polynucleotide comprising a polynucleotidesequence identical over its entire length to a coding sequence in Table1 SEQ ID NO:1, 3!. Also provided by the invention is the coding sequencefor the mature polypeptide or a fragment thereof, by itself as well asthe coding sequence for the mature polypeptide or a fragment in readingframe with other coding sequence, such as those encoding a leader orsecretory sequence, a pre-, or pro- or prepro- protein sequence. Thepolynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5' and 3' sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al.,Cell 37: 767 (1984). Polynucleotides of the invention also include, butare not limited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

A preferred embodiment of the invention is the polynucleotide comprisingnucleotide 1 to 1648 set forth in SEQ ID NO:1 of Table 1 which encodesthe proS polypeptide.

The invention also includes polynucleotides of the formula set forth inTable 1 (C) wherein, at the 5' end of the molecule, X is hydrogen, andat the 3' end of the molecule, Y is hydrogen or a metal, R₁ and R₂ isany nucleic acid residue, and n is an integer between 1 and 1000. Anystretch of nucleic acid residues denoted by either R group, where R isgreater than 1, may be either a heteropolymer or a homopolymer,preferably a heteropolymer.

The term "polynucleotide encoding a polypeptide" as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Streptococcus pneumoniae proScomprising an amino acid sequence set out in Table 1 SEQ ID NO:2, 4!.The term also encompasses polynucleotides that include a singlecontinuous region or discontinuous regions encoding the polypeptide (forexample, interrupted by integrated phage or an insertion sequence orediting) together with additional regions, that also may contain codingand/or non-coding sequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide comprisinga deduced amino acid sequence of Table 1 SEQ ID NO:2, 4!. Variants thatare fragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingproS variants, that comprising an amino acid sequence of proSpolypeptide of Table 1 SEQ ID NO:2, 4! in which several, a few, 5 to 10,1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deletedor added, in any combination. Especially preferred among these aresilent substitutions, additions and deletions, that do not alter theproperties and activities of proS.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding proS polypeptide comprising an amino acid sequence set out inTable 1 SEQ ID NO:2, 4!, and polynucleotides that are complementary tosuch polynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding proS polypeptide ofthe deposited strain and polynucleotides complementary thereto. In thisregard, polynucleotides at least 90% identical over their entire lengthto the same are particularly preferred, and among these particularlypreferred polynucleotides, those with at least 95% are especiallypreferred. Furthermore, those with at least 97% are highly preferredamong those with at least 95%, and among these those with at least 98%and at least 99% are particularly highly preferred, with at least 99%being the more preferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by the DNA of Table 1 SEQ ID NO:1!.

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms "stringent conditions" and "stringent hybridizationconditions" mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in 0.1×SSC at about 65° C. Hybridization and wash conditions are well known andexemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter11 therein.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 or 3 under stringent hybridization conditions with a probehaving the sequence of said polynucleotide sequence set forth in SEQ IDNO:1 or 3 respectively or a fragment thereof; and isolating said DNAsequence. Fragments useful for obtaining such a polynucleotide include,for example, probes and primers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding proS and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the proS gene. Such probes generally will comprise atleast 15 bases. Preferably, such probes will have at least 30 bases andmay have at least 50 bases. Particularly preferred probes will have atleast 30 bases and will have 50 bases or less.

For example, the coding region of the proS gene may be isolated byscreening using the DNA sequence provided in SEQ ID NO: 1 or 3 tosynthesize an oligonucleotide probe. A labeled oligonucleotide having asequence complementary to that of a gene of the invention is then usedto screen a library of cDNA, genornic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of SEQ ID NOS:1 and/or 2 and/or 3 and/or 4 may be used inthe processes herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, Host cells, Expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli, streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the proS polynucleotides ofthe invention for use as diagnostic reagents. Detection of proS in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of a disease. Eukaryotes (herein also"individual(s)"), particularly mammals, and especially humans, infectedwith an organism comprising the proS gene may be detected at the nucleicacid level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled proS polynucleotide sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNasedigestion or by differences in melting temperatures. DNA sequencedifferences may also be detected by alterations in the electrophoreticmobility of the DNA fragments in gels, with or without denaturingagents, or by direct DNA sequencing. See, e.g., Myers et al., Science,230: 1242 (1985). Sequence changes at specific locations also may berevealed by nuclease protection assays, such as RNase and S1 protectionor a chemical cleavage method. See, e.g., Cotton et al., Proc. Natl.Acad. Sci., USA, 85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA or cDNA may also be used for the same purpose, PCR orRT-PCR. As an example, PCR primers complementary to a nucleic acidencoding proS can be used to identify and analyze mutations. Theinvention further provides these primers with 1, 2, 3 or 4 nucleotidesremoved from the 5' and/or the 3' end. These primers may be used for,among other things, amplifying proS DNA isolated from a sample derivedfrom an individual. The primers may be used to amplify the gene isolatedfrom an infected individual such that the gene may then be subject tovarious techniques for elucidation of the DNA sequence. In this way,mutations in the DNA sequence may be detected and used to diagnoseinfection and to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStreptococcus pneumoniae, and most preferably otitis media,conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleuralempyema and endocarditis, and most particularly meningitis, such as forexample infection of cerebrospinal fluid, comprising determining from asample derived from an individual a increased level of expression ofpolynucleotide having a sequence of Table 1 SEQ ID NO: 1, 3!. Increasedor decreased expression of proS polynucleotide can be measured using anyon of the methods well known in the art for the quantitation ofpolynucleotides, such as, for example, amplification, PCR, RT-PCR, RNaseprotection, Northern blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of proS protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a proSprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. "Antibodies" as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,sirnianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various-techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, tranrsgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-proS or from naive libraries (McCafferty,J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope--termed `bispecific` antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against proS- polypeptide may be employedto treat infections, particularly bacterial infections and especiallyotitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term "antigenically equivalent derivative" as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm "immunologically equivalent derivative" as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken, The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be "humanized"; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody , for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal.,(1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem. 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS, 1986:83,9551), encapsulation of DNA in various forms ofliposomes (Kaneda et al., Science 1989:243,375), particle bombardment(Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol1993, 12:791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS 1984:81,5849).

Antagonists and Agonists--Assays and Molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of proSpolypeptides or polynucleotides, particularly those compounds that arebacteriostatic and/or bacteriocidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagonists, a synthetic reaction mix, a cellular compartment, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising proS polypeptide and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a proS agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the proS polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of proS polypeptide aremost likely to be good antagonists. Molecules that bind well andincrease the rate of product production from substrate are agonists.Detection of the rate or level of production of product from substratemay be enhanced by using a reporter system. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in proS polynucleotide or polypeptide activity,and binding assays known in the art.

Another example of an assay for proS antagonists is a competitive assaythat combines proS and a potential antagonist with proS-bindingmolecules, recombinant proS binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. proS can be labeled, such as byradioactivity or a colorimetric compound, such that the number of proSmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing proS-induced activities, thereby preventing the action of proSby excluding proS from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of proS.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock pros protein-mediated mammalian cell invasion by, for example,initiating phosphorylation of mammalian tyrosine kinases (Rosenshine etal., Infect. Immun. 60:2211 (1992); to block bacterial adhesion betweenmammalian extracellular matrix proteins and bacterial proS proteins thatmediate tissue damage and; to block the normal progression ofpathogenesis in infections initiated other than by the implantation ofin-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat otitis media, conjunctivitis, pneumonia,bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, andmost particularly meningitis, such as for example infection ofcerebrospinal fluid.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with proS, or a fragment or variantthereof, adequate to produce antibody and/ or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Streptococcus pneumoniae infection. Also providedare methods whereby such immunological response slows bacterialreplication. Yet another aspect of the invention relates to a method ofinducing immunological response in an individual which comprisesdelivering to such individual a nucleic acid vector to direct expressionof proS, or a fragment or a variant thereof, for expressing proS, or afragment or a variant thereof in vivo in order to induce animmunological response, such as, to produce antibody and/ or T cellimmune response, including, for example, cytokine-producing T cells orcytotoxic T cells, to protect said individual from disease, whether thatdisease is already established within the individual or not. One way ofadministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise. Such nucleic acid vector may compriseDNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a proS or protein coded therefrom,wherein the composition comprises a recombinant proS or protein codedtherefrom comprising DNA which codes for and expresses an antigen ofsaid proS or protein coded therefrom. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity or cellular immunity such as that arising from CTL orCD4+T cells.

A proS polypeptide or a fragment thereof may be fused with co-proteinwhich may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Streptococcus pneumoniae will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStreptococcus pneumoniae infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain proSprotein, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

Compositions, Kits and Administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStreptococcus pneumoniae wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1

Strain selection, Library Production and Sequencing

The polynucleotide having the DNA sequences given in SEQ ID NO:1 and 3were obtained from a library of clones of chromosomal DNA ofStreptococcus pneumoniae in E. coli. The sequencing data from two ormore clones containing overlapping Streptococcus pneumoniae DNAs wasused to construct the contiguous DNA sequence in SEQ ID NO:1 and 3.Libraries may be prepared by routine methods, for example: Methods 1 and2 below.

Total cellular DNA is isolated from Streptococcus pneumoniae 0100993according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E.coli infected with the packagedlibrary. The library is amplified by standard procedures.

Example 2

ProS Characterization

The enzyme mediated incorporation of radiolabelled amino acid into tRNAmay be measured by the aminoacylation method which measures aminoacid-tRNA as trichloroacetic acid-precipitable radioactivity fromradiolabelled amino acid in the presence of tRNA and ATP (Hughes J,Mellows G and Soughton S, 1980, FEBS Letters, 122:322-324). Thusinhibitors of prolyl tRNA synthetase can be detected by a reduction inthe trichloroacetic acid precipitable radioactivity relative to thecontrol. Alternatively the tRNA synthetase catalysed partial PPi/ATPexchange reaction which measures the formation of radiolabelled ATP fromPPi can be used to detect prolyl tRNA synthetase inhibitors (Calender R& Berg P, 1966, Biochemistry, 5, 1681-1690).

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 4    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1651 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: Genomic DNA    -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:1:    #TGCTCAAGTT    60AAATGCC TATCCCAACG CTTCGCGAAA TGCCAAGCGA    #AGGTGTTTAT   120TTATGTT GCGTGCTGGT TATGTTCGCC AAGTTTCAGC    #GCGCCAAGAA   180TTGCCAA CCGTGTGATT GAAAAAGCTA AAAACATCAT    #AGAATTGTGG   240GTGCTGT TGAGATGTTG GCTCCAGCCC TTCTTAGTGC    #AAACCGTGAA   300GTTACGA AACCTATGGT GAAGACCTTT ACAAACTGAA    #TGTCCGTGAT   360TCTTAGG TCCAACTCAC GAAGAAACCT TTACAGCTAT    #CAAGTATCGT   420ACAAGCA ATTGCCACTC AACCTTTATC AAATTCAGCC    #GAAGGATGCT   480CACGTAA TGGACTTCTT CGTACACGTG AGTTTATCAT    #CAAAGCAGCC   540CTAACTA TGATAGTTTG GATAGTGTTT ATGATGAGTA    #TGACGGTGGA   600TCACTCG TAGTGGTTTA GACTTCAAGG CTATTATTGG    #CCGTACAGAC   660AGGATAG CCAAGAATTT ATGGCCATTA GGCCTATCTG    #TCCTGCAGAA   720TTGTCTT GGACAAGTCA GTTGCCTCAT TTGACGAAAT    #AGATACCATT   780TCAAGGC AGAATTGCTC AAATGGATAG TCTCTGGTGA    #CGAGTACAAA   840AGTCTAG CTATGCAGCT AACTTAGAAA TGGCAACAAA    #AGATGTTAAA   900TTGTCGC TGAAGAAGAA GTTACTCGTG TTGAAACGCC    #TAAAACCCTC   960TTGCAGC CTTCCTCAAT GTTCCAGAAG AACAAACGAT    #CCAACTCAAC  1020ATGGTGA GCTTGTTGCA GCCCTTCTAG TTGGAAATGA    #CGAAGAAGAA  1080AAAATCA CTTGGGAGCA AATTTCTTTG ACGTTGCTAG    #GCCAGAGAAT  1140TTCAAGC AGGATTTGGT TCACTTGGAC CAGTTGGTTT    #CGGTGCTAAC  1200CAGATCG TAAGGTGCAA GATGTTCGCA ATGCAGTTGT    #AGAATATGTG  1260ACTTGAC TGGTGTGAAC CCAGGCCGTG ATTTTACTGC    #CCTTAACTTT  1320TTCGTGA GGGTGAAATT TCCCCAGATG GACAAGGTGT    #AGCAAGCATG  1380AGATCGG TCATATTTTC AAACTCGGAA CTCGCTATTC    #ATGTTACGGT  1440TGGATGA AAATGGTCGT GCTGTGCCAA TCATCATGGG    #CTTTGTTAAC  1500GTCTTCT TTCAGCAGTG ATGGAGCAAC ACGCTCGCCT    #AGAATTGGCA  1560GTGAATA CCGTTACGCT TGGGGAATCA ATTTCCCTAA    #AGCCTTGACA  1620ATTTGAT TACTGTTAAT GTCAAGGATG AAGAAGCGCA    #        1651      AGCTT GATGGGAGAT C    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 550 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:2:    #Leu Arg Glu Met Pro Seret Pro Ile Pro Thr    #                 15    #Leu Arg Ala Gly Tyr Valer His Ala Leu Met    #             30    #Leu Pro Leu Ala Asn Argly Val Tyr Ser Tyr    #         45    #Gln Glu Phe Glu Lys Ileys Asn Ile Met Arg    #     60    #Leu Ser Ala Glu Leu Trpeu Ala Pro Ala Leu    # 80    #Glu Asp Leu Tyr Lys Leuyr Glu Thr Tyr Gly    #                 95    #Gly Pro Thr His Glu Gluer Asp Phe Ile Leu    #            110    #Lys Ser Tyr Lys Gln Leual Arg Asp Ser Val    #        125    #Tyr Arg Asp Glu Lys Argln Ile Gln Pro Lys    #    140    #Phe Ile Met Lys Asp Alaeu Arg Thr Arg Glu    #160    #Asp Ser Val Tyr Asp Glusn Tyr Asp Ser Leu    #                175    #Arg Ser Gly Leu Asp Phelu Arg Ile Phe Thr    #            190    #Gly Gly Lys Asp Ser Glnsp Gly Gly Ala Met    #        205    #Thr Asp Leu Asp Arg Trprg Pro Ile Cys Arg    #    220    #Asp Glu Ile Pro Ala Gluer Val Ala Ser Phe    #240    #Lys Trp Ile Val Ser Glyys Ala Glu Leu Leu    #                255    #Ser Tyr Ala Ala Asn Leuyr Ser Ser Glu Ser    #            270    #Asn Arg Val Val Ala Glulu Tyr Lys Pro Ser    #        285    #Val Lys Ser Ile Asp Glual Glu Thr Pro Asp    #    300    #Gln Thr Ile Lys Thr Leusn Val Pro Glu Glu    #320    #Ala Leu Leu Val Gly Asnly Glu Leu Val Ala    #                335    #His Leu Gly Ala Asn Pheal Lys Leu Lys Asn    #            350    #Asn Val Val Gln Ala Glylu Glu Glu Val Ala    #        365    #Glu Asn Ile Lys Ile Ilero Val Gly Leu Pro    #    380    #Ala Val Val Gly Ala Asnln Asp Val Arg Asn    #400    #Pro Gly Arg Asp Phe Threu Thr Gly Val Asn    #                415    #Glu Gly Glu Ile Ser Prole Arg Glu Val Arg    #            430    #Gly Ile Glu Ile Gly Hiseu Asn Phe Ala Arg    #        445    #Ser Met Gly Ala Asp Valhr Arg Tyr Ser Ala    #    460    #Ile Met Gly Cys Tyr Glyrg Ala Val Pro Ile    #480    #Met Glu Gln His Ala Argeu Leu Ser Ala Val    #                495    #Tyr Arg Tyr Ala Trp Glyhr Pro Lys Gly Glu    #            510    #Asp Val His Leu Ile Thrlu Leu Ala Pro Phe    #        525    #Leu Thr Glu Lys Leu Glulu Glu Ala Gln Ala    #    540    -  Ala Ser Leu Met Gly Asp    #550    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 487 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: Genomic DNA    -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:3:    #TGTTGAGATG    60AAAACAT CATGCGCCAA GAATTCGAAA AGATTGGTGC    #CGAAACGTAT   120TTCTTAG TGCAGAATTG TGGCGTGAAT CAGGTCGTTA    #AGGTCCAACT   180ACAAACT GAAAAACCGT GAAAAATCAG ACTTTATCTT    #GCAATTGCCA   240TTACAGC TATTGTCCGT GATTCTGTTA AATCTTACAA    #TAATGGACTT   300AAATTCA GCCCAAGTAT CGTGATGAAA AACGCCCACG    #CTATGATAGT   360AGTTTAT CATGAAGGAT GCTTATAGTT TCCACGCTAA    #TCGTAGTGGT   420ATGATGA GTACAAAGCA GCCTATGAGC GTATTTTCAC    #TAGCCAAGAA   480CTATTAT TGGTGACGGT GGAGCCATGG GTGGTAAGGA    #         487    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 162 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -            (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:4:    #Glu Phe Glu Lys Ile Glysn Ile Met Arg Gln    #                 15    #Ser Ala Glu Leu Trp Argla Pro Ala Leu Leu    #             30    #Asp Leu Tyr Lys Leu Lyslu Thr Tyr Gly Glu    #         45    #Pro Thr His Glu Glu Thrsp Phe Ile Leu Gly    #     60    #Ser Tyr Lys Gln Leu Prorg Asp Ser Val Lys    # 80    #Arg Asp Glu Lys Arg Prole Gln Pro Lys Tyr    #                 95    #Ile Met Lys Asp Ala Tyrrg Thr Arg Glu Phe    #            110    #Ser Val Tyr Asp Glu Tyryr Asp Ser Leu Asp    #        125    #Ser Gly Leu Asp Phe Lysrg Ile Phe Thr Arg    #    140    #Gly Lys Asp Ser Gln Gluly Gly Ala Met Gly    #160    -  Phe Met    __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide segment encoding SEQID NO:
 2. 2. An isolated nucleic acid segment comprising a nucleotidesequence which is fully complementary to the polynucleotide of claim 1.3. An isolated vector comprising the polynucleotide segment of claim 1.4. An isolated vector comprising the nucleic acid segment of claim
 2. 5.An isolated host cell comprising the vector of claim
 3. 6. An isolatedhost cell comprising the vector of claim
 4. 7. A process for producing apolypeptide encoded by said polynucleotide segment comprising culturingthe host cell of claim 5 under conditions sufficient for the productionof said polypeptide.
 8. An isolated polynucleotide segment encoding SEQID NO:
 4. 9. An isolated nucleic acid segment comprising a nucleotidesequence which is fully complementary to the polynucleotide of claim 8.10. An isolated vector comprising the polynucleotide segment of claim 8.11. An isolated vector comprising the nucleic acid segment of claim 9.12. An isolated host cell comprising the vector of claim
 10. 13. Anisolated host cell comprising the vector of claim
 11. 14. A process forproducing a polypeptide encoded by said polynucleotide segmentcomprising culturing the host cell of claim 12 under conditionssufficient for the production of said polypeptide.
 15. An isolatedpolynucleotide segment encoding a mature polypeptide expressed by apolynucleotide comprising SEQ ID NO: 1 in deposited strain NCIMB 40794or NCIMB
 40800. 16. An isolated nucleic acid segment comprising anucleotide sequence which is fully complementary to the polynucleotidesegment of claim
 15. 17. An isolated vector comprising thepolynucleotide segment of claim
 15. 18. An isolated vector comprisingthe nucleic acid segment of claim
 16. 19. An isolated host cellcomprising the vector of claim
 17. 20. An isolated host cell comprisingthe vector of claim
 18. 21. A process for producing the maturepolypeptide comprising culturing the host cell of claim 19 underconditions sufficient for the production of said polypeptide.